Somatic hypermutation of variable genes, which encode a portion of immunoglobulin molecules, occurs at a frequency that is a million times greater than mutation in other genes. The molecular mechanism that introduces these mutations is intensely being studied. Hypermutation is initiated when the activation-induced cytosine deaminase protein deaminates cytosine in DNA to uracil, and the uracil lesion is inaccurately repaired by a DNA polymerase. This predicts that variable genes should have an abundance of uracils. We are currently measuring levels of uracil in DNA from mutating cell lines. To see if a lesion of guanine also initiates the process, we examined hypermutation in mice deficient for 8-hydroxyguanine-DNA glycosylase, an enzyme that removes oxidized guanine from DNA. The frequency of mutation and types of nucleotide substitutions were similar to wildtype mutations. The findings show that modified guanines do not cause variable gene mutations. To study the roles of DNA polymerases in introducing mutations during repair of the uracil lesion, we have examined two low-fidelity enzymes, eta and iota. For polymerase eta, variable genes were sequenced from patients who are deficient in the polymerase. The frequency of hypermutation was normal, but the types of base changes were different. Polymerase eta-deficient clones had a decrease in the proportion of mutations at A and T with a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation. For polymerase iota, variable genes were sequenced from mice that are deficient in the polymerase. The frequency and pattern of hypermutation was normal. Thus, either polymerase iota does not participate, or its role is non-essential and can be assumed by polymerase eta. Mismatch repair proteins also affect the pattern of hypermutation, indicating that they participate in the mechanism. To test for the role of MSH2, MSH3, and MSH6 in the process, we are using various biochemical techniques to identify proteins that interact with the repair molecules. It will soon be possible to assemble most of the pieces that make up the enigmatic hypermutation puzzle.